Effect of Salicylic Acid Foliar Application on Bioactive Compounds and Antioxidant Activity in Holy Basil ( Ocimum sanctum L.)

Holy basil ( Ocimum sanctum L.) has been used extensively in Tai traditional medicine, where it is commonly utilized as a part of herbal remedies for treating various ailments. Cultivation methods using exogenous salicylic acid (SA) to induce secondary metabolites have been documented in various plant species. Nevertheless, there is no reported information available on holy basil. Tus, the present study aimed to investigate the impact of SA foliar application on the bioactive compounds and antioxidant activity of holy basil. SA at concentrations of 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5mM was foliar sprayed 30days after transplanting (DAT) compared to spraying with tap water as the control. Te plants were harvested at 33 DAT. Exogenous SA at 0.1–1.5mM enhanced the contents of bioactive compounds and improved antioxidant activity. Te highest contents of eugenol (17,829.53 ± 243.11 μ g/g dry extract), total phenolics (444.10 ± 2.80mg GAE/g dry extract), and total favonoids (382.69 ± 6.49mg QE/g dry extract) were achieved at 1.0mM SA foliar application, which was 282.96, 1.76, and 2.14 times, respectively, over control. Furthermore, the greatest antioxidant activity was observed in the 1.0mM SA treatment. In contrast, the 2.0 and 2.5mM SA treatments had lower levels of antioxidant activity and bioactive compounds than the control. Te results of this study suggest that exogenous 1.0mM SA foliar application is an efective method to produce enriched bioactivity in holy basil


Introduction
Holy basil (Ocimum sanctum L.) is one of the most popular medicinal plants in the Lamiaceae family.Since ancient times, this plant species has been extensively employed in traditional medicine to prevent and treat ailments [1].Its leaves are rich in eugenol, methyl eugenol, phenolics, favonoids, terpenoids, neolignans, and fatty acid derivatives [2].Tese constituents possess pharmacological properties that modulate various biological activities, including antioxidant, anticancer, antiasthmatic, anti-infammatory, antiallergic, antidiabetic, and antistress [1][2][3].In Tai traditional medicine, holy basil leaves are used in herbal remedies for treating cancer as well as relieving fatulence and asthma.
Components derived from plants, such as roots, shoots, leaves, fowers, fruits, and seeds, play a crucial role as fundamental elements in creating herbal products.Te high content of bioactive compounds in medicinal plants is regarded as a high-quality raw material for the herbal drug industry.Te accumulation of secondary metabolites in these plants can be induced by several cultivation methods [4].Biotic elicitors are commonly applied to enhance the synthesis and accumulation of bioactive compounds in plants since it is convenient, fast, safe, eco-friendly, and quick strategy [5].A highly efective biotic elicitor known as salicylic acid (SA) or 2-hydroxybenzoic acid causes a variety of physiological and developmental responses in plants, including seed germination [5,6], stomatal movement [5,6], pigment accumulation [5,7], photosynthesis [5,6,8], ethylene biosynthesis [5], enzyme activities [5], nutrient uptake [5,8], fower induction [5,6], and membrane functions [5].Furthermore, SA has been increasingly recognized for its role in enhancing plant tolerance to various abiotic stresses such as salinity, drought, and high temperature [9].Exogenous SA has been applied to increase bioactive compounds in many plant species such as sweet basil [10][11][12], peppermint [8,13], marjoram [10], amaranth [14], and Ammi visnaga [15].Te efciency of exogenous SA to induce secondary metabolites depends on many factors.SA concentration is one of the important factors infuencing bioactive compounds in plant species [16].Low SA concentration might be not enough for physiological process activation or to regulate gene expression for secondary metabolite accumulation [17].In contrast, high SA concentration usually causes deleterious efects.However, the optimal SA concentration to induce bioactive compounds in each plant species tends to be specifc, like 0.1 mM SA for sweet basil [12], 0.01 mM SA for amaranth [14], 0.5 mM SA for peppermint [8], and 1 and 2 mM for Ammi visnaga under water shortage and normal irrigation, respectively [15].Tese values indicate that each plant species shows diferent responses to SA doses.However, no information has been reported on holy basil.Terefore, the purpose of this work was to examine the eugenol content as well as the total phenolic, favonoid, and antioxidant activity in holy basil at various exogenous SA application concentrations.Te fndings of this study will provide technical guidance for the production of enriched bioactivity in holy basil.

Plant Cultivation, Salicylic Acid Treatment, and Plant
Growth Measurement.Holy basil, purple-type seeds of OS18 were collected at Tammasat University, Tailand.Te seeds were sown into 105-cell trays containing peat moss as substrate.Te 30-day-old seedlings were transplanted into 8 × 15-inch planting bags.Te physical and chemical properties of the commercial substrates, as described by Rithichai et al. [18], were utilised in this study.Te base fertilizer consisted of 30 g/plant of manual fertilizer and 0.65 g/plant of 16-16-16 (N-P-K).After transplanting, the plants were fertilised with a solution containing 6.5 g/L urea at a rate of 100 ml/plant for 7 days.At 15 days after transplanting (DAT), the plants were further fertilised with 0.65 g/plant of 16-16-16.Water was irrigated daily to maintain soil moisture.Te plants were cultivated in a greenhouse located at Tammasat University, Rangsit Campus, Pathum Tani, Tailand.Plants at 30 DAT were foliar sprayed with SA at the concentrations of 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mM for 100 mL/plant.Te control was sprayed with tap water.Tween 20 at 0.1 mL/L was used to help spread and keep the spray solution on the foliage.Plant height was measured at 33 DAT, then the plants were cut at the soil surface, and shoot fresh weight was recorded.Mature leaves were separated, and leaf fresh weight was collected.Leaves were dried by freeze drier, and leaf dry weight was determined.Te freeze-dried samples were ground and kept at −20 °C for further use.

Preparation of Alcoholic
Extracts.Plant extract samples were prepared as described by Rithichai et al. [18].After calculating the dry extract's yield, samples of the dried extract were stored at −20 °C for later use.

Determination of Eugenol Content.
Eugenol content was conducted following the method described by Autaijamsripon et al. [19] with some modifcations.Ten milligrams of the extract were dissolved with 1 mL of HPLC methanol, and the solution was sonicated for 20 min.Each sample was fltered through a 0.22 μm flter membrane.To determine the eugenol content, ultrahigh performance liquid chromatography (UHPLC) was used.Te Nova-Pak C18 column (150 × 3.9 mm i.d., 4 μm) with a guard column was connected to the Shimadzu Nexera LC-30 A with isocratic elution mode.Ten microliters of the sample were injected for 4 min.Acetonitrile, water, and methanol were combined in the mobile phase in a ratio of 50 : 40 : 10.Tere was a 0.7 ml/ min fow.Te wavelength of the detector was tuned to 280 nm (Figure 1).Eugenol content was calculated using a lab solution program and expressed as μg/g dry extract.

Determination of Total Phenolic and Flavonoid Contents.
Total phenolic and favonoid contents were determined using the Folin-Ciocalteu colourimetric method [18] and aluminium chloride colourimetric assays [20], respectively.To examine the total phenolic and favonoid contents, a microplate reader (Power Wave XS, BioTek) was utilised to measure the absorbance at wavelengths of 765 and 510 nm, respectively.Total phenolic content was expressed as milligram gallic acid equivalent per gram dry extract (mg GAE/ g dry extract), while the total favonoid content was expressed as milligram quercetin equivalent per gram dry extract (mg QE/g dry extract).

Determination of Antioxidant Activity.
Antioxidant activity was measured using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, employing a method adapted from Rithichai et al. [18].Absorbance was measured at 520 nm using a microplate reader.EC 50 values, which represent the concentration of the sample needed to inhibit 50% of the DPPH free radical, were calculated using a regression equation.Te positive control was BHT (butylated hydroxytoluene).

Statistical Analysis.
Te experiment employed a completely randomized design.Data were subjected to analysis of variance (ANOVA) using SAS software.Diferences between means were achieved by Tukey's Honestly Signifcant 2 International Journal of Agronomy Diference (HSD) at P < 0.05.Te mean values of three replicates were presented.Te correlation between SA concentration, bioactive compounds, and antioxidant activity in the holy basil leaves was assessed using Pearson's correlation test, while principal component analysis (PCA) was carried out using JMP statistical software.SA, an endogenous regulator, may have an impact on plant growth and production [21].It stimulates plant growth by changing the hormonal status and improving photosynthesis, transpiration, and stomatal conductance [6,8].Te efect of exogenous SA on growth depends on various factors, such as plant species, developmental stages, and concentration.Plant growth of holy basil in the present study was not infuenced by SA foliar application at diferent doses.Moreover, a single application might not be sufcient to stimulate mechanisms for plant growth.Similar results were reported by Damalas [22] who stated that under 100% feld capacity, there were no signifcant diferences between 200 ppm SA foliar application (applied once at 5-6 true leaves stage) and control for sweet basil shoot fresh weight, dry weight, or plant height.Jaafari and Hadavi [11] showed that foliar spray of SA three times (at 24, 34, and 44 days after emergence) at the concentrations of 2 and 4 mM did not afect the fresh weight and dry weight of sweet basil.In contrast, exogenous SA application increased plant growth of sweet basil [10,23], marjoram [10], peppermint [8], and Ammi visnaga [15].Elhindi et al. [23] showed that the application of 1.0 mM SA twice, frst at the seedling stage with 2-3 true leaves and then 5 days after transplanting, signifcantly increased plant height, shoot fresh and dry weight, branch number, and leaf area of sweet basil.Likewise, Gharib [10] reported that 10 −4 M SA foliar application at 75 and 82 days after sowing, with a repeat two weeks after the frst and second cut, stimulated the growth of sweet basil and marjoram by enhancing photosynthesis and nutrient uptake.Te 2.0 mM SA treatment resulted in improvement of the leaves and shoots in peppermint [8].Te application of similar concentrations of SA (2 mM) seven times increased the plant growth of Ammi visnaga under normal irrigation [15].Tese fndings indicate that the enhancement of plant growth by SA depends not only on its concentrations but also on the frequency of applications.

Efect of SA Concentrations on Bioactive Compounds.
Te yield of the dry extract ranged from 17.01 ± 3.47 to 20.81 ± 2.50%, and there were no signifcant diferences observed among the SA foliar applications at various concentrations (Figure 3).
SA foliar application at various concentrations afected the eugenol content in holy basil.Te maximum eugenol content of 17,829.53± 243.11 μg/g dry extract occurred in 1.0 mM SA treatment, which increased 282.96 times over control.Eugenol content exhibited signifcant increases of International Journal of Agronomy 68.64, 182.02, and 78.21 times above control when applied SA at the concentrations of 0.1, 0.5, and 1.5 mM, respectively.Low eugenol contents of 201.09 ± 12.98 and 244.21 ± 12.75 μg/g dry extract were obtained in 2.0 and 2.5 mM SA treatments, respectively, which were not signifcantly diferent compared to the control (Figure 4(a)).SA concentrations of 0.1, 0.5, 1.0, and 1.5 mM induced the accumulation of total phenolics.Te highest content of total phenolics of 444.10 ± 2.80 mg GAE/g dry extract was observed in 1.0 mM SA treatment which was 1.76 time higher than control.While 2.0 and 2.5 mM SA treatments resulted in a lower total phenolic content of 209.01 ± 3.69 and 189.99 ± 1.35 mg GAE/g dry extract, respectively, than the control (Figure 4(b)).
Te changes in total favonoid contents under SA foliar application exhibited a similar trend as total phenolic contents.Te maximum content of total favonoids of 382.69 ± 6.49 mg QE/g dry extract occurred in 1.0 mM SA treatment which was 2.14 times above control.Total favonoid contents of 0.1, 0.5, and 1.5 mM SA treatments showed higher values than those of the control while those of 2.0 and 2.5 mM SA treatments exhibited lower values of 140.42 ± 2.91 and 129.67 ± 5.97 mg QE/g dry extract, respectively, than those of the control (Figure 4(c)).
SA concentrations are crucial in inducing holy basil to produce bioactive compounds.In the present study, low SA concentrations at 0.1, 0.5, 1.0, and 1.5 mM demonstrated efective induction of eugenol and total phenolic and favonoid accumulation.A maximum increase was observed at 1.0 mM SA treatment as the contents of eugenol, total phenolics, and total favonoids were 282.96, 1.76, and 2.14 times, respectively, above control.On the contrary, high SA concentrations at 2.0 and 2.5 mM showed negative efects as the contents of those bioactive compounds were lower than those of control.Tese results indicated that SA stimulated bioactive compound accumulation in holy basil depending on its  International Journal of Agronomy concentration.As a hormone-like substance of SA, high concentrations are typically harmful to physiological processes and can cause adverse efects, whereas appropriate SA concentrations regulate key enzymes like phenylalanine ammonia lyase and isochorismate synthase, which promote the formation of secondary metabolites and their subsequent storage in plant tissue [5].Te fndings of this study were in agreement with those of earlier research, as exogenous SA application at low doses signifcantly induced the accumulation of bioactive compounds [8,10,11,13].Figueroa-Pérez et al. [8] reported that foliar application of 0.5 and 1.0 mM SA was more efective for enhancing total phenolic and favonoid contents in peppermint than 2.0 mM SA. Essential oil content of peppermint was signifcantly increased at 150 mg/L SA treatment, but it was statistically decreased under increasing SA concentrations of 300 and 400 mg/L treatments [13].Similar to sweet basil, the exogenous application of 2.0 mM SA resulted in a higher yield of essential oil compared to 4.0 mM SA [11].Moreover for sweet basil, the higher eugenol level [10] and essential oil content [12] over control were obtained in foliar spray SA at 1.0 mM.

Efect of SA Concentrations on Antioxidant Activity.
Te strongest antioxidant activity with the lowest EC 50 value of 7.49 ± 1.00 μg/mL was achieved in 1.0 mM SA treatment.Te EC 50 value of BHT, the positive control, was 11.24 ± 0.63 μg/mL.Te 0.1, 0.5, and 1.5 mM SA treatments also revealed lower EC 50 values than the control.On the other hand, the weak antioxidant activities occurred in 2.0 and 2.5 mM SA treatments, in which EC 50 values of 19.73 ± 0.72 and 22.36 ± 1.11 μg/mL, respectively, were higher than those of the control (Figure 4(d)).
Antioxidant measured by a DPPH radical scavenging assay is commonly used to determine the ability of the sample to provide hydrogen atoms.Te response of antioxidant activity to exogenous SA concentrations exhibited the same trend as bioactive compounds.Low SA concentrations between 0.1 and 1.5 mM showed a signifcant decrease in EC 50 values in comparison to the control.Te 1.0 mM SA showed the most efective antioxidant activity as the lowest EC 50 value occurred.Tese efects could contribute to the increase in bioactive compound accumulation in low SA concentration treatments.Eugenol, total phenolics, and total favonoids are the major antioxidant compounds in holy basil [2].Tese compounds revealed a strong negative correlation with EC 50 values of antioxidant activity as shown in Table 1 (R � −0.826, R � −0.940, and R � −0.922, respectively, P < 0.01).Tis implied that a high content of these antioxidant compounds had the potential to scavenge free radicals.Antioxidant activities responded to SA doses were diferent among plant species.Exogenous SA at low doses, known for its potent antioxidant activity, has been documented in other plant species.Te infusions prepared from peppermint leaves treated with 0.5 and 1.0 mM SA exhibited a greater capacity to inhibit DPPH radicals compared to those treated with 2.0 mM SA treatment [8].

Correlation Analysis and PCA of SA Concentration,
Bioactive Compounds, and Antioxidant Activity.Te SA concentration exhibited a moderate negative correlation with total favonoid content, while showing a strong positive correlation with the EC 50 value of antioxidant activity (Table 1).Moreover, the SA concentration displayed no International Journal of Agronomy correlation with eugenol and total phenolic content.Te results suggested that the application of exogenous SA at low concentrations resulted in increased levels of total favonoids and greater antioxidant activity.However, it did not have any efect on eugenol and total phenolic content.PCA was conducted to enhance the comprehension of correlations among the variables.Te initial two principal components, PC1 and PC2, explained 97.9% of the total variation (Figure 5).PC1 was signifcantly infuenced by all variables.Notably, the vectors representing SA concentration and antioxidant activity were opposite to those of eugenol, total phenolic, and total favonoid contents on PC1, indicating an absence of direct association between bioactive compounds and SA concentration as well as antioxidant activity.All variables loaded positively on PC2, with antioxidant activity and total phenolic and favonoid contents being less signifcant compared to SA concentration and eugenol.Te samples with identical SA concentrations formed distinct clusters.Specifcally, samples with SA concentrations of 0.1, 0.5, 1.0, and 1.5 mM exhibited positive loading on PC1, indicating higher contents of eugenol and total phenolic and total favonoid compounds, coupled with lower levels of EC 50 of antioxidant activity.Conversely, SA concentrations of 0, 2.0, and 2.5 mM exhibited negative loading on PC1, indicating lower content of bioactive compounds and higher levels of EC 50 of antioxidant activity.
To accelerate bioactive compound accumulation, plants usually respond to the elicitor during a short duration after application.However, the appropriate preharvest application time inducing secondary metabolites difered in each plant species.For example, the 0.01 mM SA exogenous applied at 1 day before harvest revealed the highest total phenolic content in coriander [24].In the case of Chinese kale, higher contents of vitamin C, total chlorophyll, and total phenolics occurred in 1.0 mM SA foliar sprayed at 6 days before harvest [25] while mustard foliar sprayed with 10 −2 mM at 15 days before harvest resulted in an increase of total chlorophyll peroxidase and superoxide dismutase [26].Terefore, we continuously studied the preharvest times of exogenous 1.0 mM SA application at 1, 2, 3, 4, and 5 days before harvest.It was found that SA foliar sprayed three days before harvest possessed the highest contents of bioactive compounds and antioxidant activity in holy basil (data not shown).Based on the current study, exogenous SA application could be a promising tool to enhance bioactivity in holy basil.Te most efective method to improve the contents of eugenol, total phenolics, and total favonoids as well as antioxidant activity was 1.0 mM SA foliar sprayed three days before harvest.

Conclusions
Exogenous SA at low concentrations of 0.1-1.5 mM enhanced the contents of eugenol, total phenolics, and total favonoids in holy basil.Te strong antioxidant activity was achieved when 0.1-1.5 mM SA was foliar sprayed.Foliar application of 1.0 mM SA was an efective method to produce enriched bioactivity in holy basil.

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Figure 2 :Figure 3 :
Figure 2: Efect of SA foliar application on (a) plant height, (b) shoot and leaf fresh weight (FW), and (c) leaf dry weight (DW) of holy basil.Data are means of three replicate samples and error bars indicate ± SD.Te same uppercase letters indicate nonsignifcant diference by Tukey's HSD (honestly signifcant diference) test at P < 0.05.

Figure 4 :
Figure 4: Efect of SA foliar application on (a) eugenol content, (b) total phenolic content (TPC), (c) total favonoid content (TFC), and (d) antioxidant activity of holy basil.Data are means of three replicate samples and error bars indicate ± SD.Te same uppercase letter indicates nonsignifcant diference by Tukey's HSD (honestly signifcant diference) test at P < 0.05.

Table 1 :
Pearson's correlation coefcient between concentrations of SA, bioactive compounds, and antioxidant activity in the leaves of holy basil.